Рибосома как сенсор синтезируемого пептида и малых молекул Александр Манькин Center for Pharmaceutical Biotechnology University of Illinois at Chicago The ribosome is an amazing RNA machine Ada Yonath Tom Steitz Alexander Spirin Harry Noller, Marat Yussupov, Gulnara Yussupova Venki Ramakrishnan Elongation Initiation Termination Schmeing)&)Ramakrishnan)(2009)))Nature,)461,)1234)) pep)dyl0transferase'' center' nascent'pep)de'' exit'tunnel' Nissen)et)al.,)(2000))Science,)289,)920;)Voorhees)et)al.,)(2009))Nat.Struct.Mol.Biol.,)16,)528;))Voss et al., (2006) J Mol Biol, 360, 893 Nissen)et)al.,)2000)Science)289,920) Nakatogawa)&)Ito,)(2002))Cell,)108,629;)Gong)&)Yanofsky,)(2002))Science,)297,)1864;))Chiba)et)al.,)(2009))EMBO)J.)28,)3461;)Onouchi)et)al.(2005))Genes)Dev.)15,)1799)) Ribosome stalling controls expression of important bacterial and eukaryotic genes Regulatory ORF Regulated ORF cofactor cellular signal secM mifM XBP1u tnaC CGS1 AAP ermC Nakatogawa)&)Ito,)(2002))Cell,)108,629;)Gong)&)Yanofsky,)(2002))Science,)297,)1864;))Chiba)et)al.,)(2009))EMBO)J.)28,)3461;)Onouchi)et)al.(2005))Genes)Dev.)15,)1799)) Nissen)et)al.,)(2000))Science,)289,)920;)Voorhees)et)al.,)(2009))Nat.Struct.Mol.Biol.,)16,)528;))Voss et al., (2006) J Mol Biol, 360, 893 Erythromycin (ERY) Ketolides:Telithromycin (TEL) O N N N O O OH HO N OH O O O O O O O O O O O O HO N OH O N O O Schlunzen,)(2001))Nature,)413,814;)Tu)et)al.,)(2005))Cell,)121,)257;))Bukley)et)al.,)(2010).)PNAS)107,)17158;)Dunkle)et)al.(2010))PNAS)107,)17152)) Erm methyltransferase A2058 CH3 H3C NH2 N N H N N N N N H N N Translation attenuation controls the expression of erm genes Leader ORF Spacer 3 I'I'I'I'I'I'I'I' ermCL 2 I'I'I'I'I'I'I'I' 1 Resistance Gene 4 ermC Non-Induced 2 3 I'I'I'I'I'I'I'I' I'I'I'I'' 1 ermCL ermCL 1 I'I'I'I'I'I'I'I' 2 4 ermC 3 4 ermC Induced Horinouchi)&)Weisblum)(1980))PNAS)77,)7079;)Gryczan)et)al.(1980))Nucl)Acids)Res)8,)6081) ermCL 16-17 nt Ery C C U - + P site ermCL M G I F S I F V I S T V H Y … Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190) Specific sequence of the nascent peptide controls stalling key sequence 'MGIFSIFVI'' Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190;)Ramu)et)al.)(2011))Mol.)Cell)41,)321)) tunnel A2062 the most flexible residue in the tunnel The mutant ribosomes are functionally active, but do not ‘recognize’ the nascent peptide! Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190;)Vazquez\Laslop,)et)al.)(2010))EMBO)J.)29,)3108) Voorhees)et)al.,)(2009))NSMB,)16,)528;)Scheming)et)al.)(2005))Nature,)438,)520;)) A2062 and A2503 are sensors of the stalling signal in the tunnel A2503 ERY A2062 peptide Modified nucleotides in 23S rRNA m2A2503 NH2 N N HO NH2 N N N RlmN N O H H H OH N HO O H N OH H H OH OH H H Toh)et)al.)(2008))RNA,)14,)98)) Posttranscriptional modification of the rRNA sensor contributes to the recognition of the stalling signal ermCL wt' ermC rlmN)0' inducible'ermC' Vazquez\Laslop,)et)al.)(2010))EMBO)J.)29,)3108) A2058 Erm methyltransferase A2058 CH3 H3C NH2 N N H N N N N N H N N Modification of A2058 in the tunnel affects translation of several proteins and reduces cell fitness PoxA poxA Asn118 Gupta)et)al.)(2013),)Nature)Communica_ons,)in)press) Where do sensors send the signal? What becomes broken in the stalled ribosome? peptidyl transferase center A2503 ERY A2062 peptide ermCL M''G''I''F''S''I''F''V''I'S''T''V''H''Y''Q''P''N''K'' ERY' Ribosomal' sensors' I" V" F" I" S" F" I" G" fM" Pep)dyl' puromycin' transferase' center' ???' Stalled ribosome is unable to catalyze peptide bond formation Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190) Peptide and antibiotic affect the peptidyl transferase A-site Peptidyl transferase center is the target of the stalling signal Ramu)et)al.)(2011))Mol.)Cell)41,)321) A'site' C2452' A2503 A2062 ERY U2504' A2503 P'site' A2451' G2061' A2062 peptide The'A2062'and'A2503'sensor'in'the'exit'tunnel'may'relay'the'stalling'signal'to'the'A0site'of'the' pep)dyl'transferase'center' Translation attenuation control requires the antibiotic Leader ORF Spacer 3 I'I'I'I'I'I'I'I' ermCL 2 I'I'I'I'I'I'I'I' 1 Resistance Gene 4 ermC Non-Induced 2 3 I'I'I'I'I'I'I'I' I'I'I'I'' 1 ermCL ermCL 1 I'I'I'I'I'I'I'I' 2 4 ermC 3 4 ermC Induced Horinouchi)&)Weisblum)(1980))PNAS)77,)7079;)Gryczan)et)al.(1980))Nucl)Acids)Res)8,)6081) What is the role of the antibiotic in nascent peptide-dependent ribosome stalling? 2 3 ermCL I'I'I'I'I'I'I'I' I'I'I'I'' 1 !!! A2503 A2062 A road-block 4 ermC ERYTHROMYCIN' CLARYTHROMYCIN' O TELITHROMYCIN' O OH HO O O O O O O O O O O HO OH O O OH O O O O O O O O O O O HO OH O O O O O HO OH O O O O O HO N O O ITR163' O O OH O ITR162' N N O ITR156' O N HO OH ITR166' O O O O O O O HO N OH O HO N OH O O O N O O N OH HN O N OH HN O N OH HN O N O O Vázquez\Laslop)et)al.)(2011),)PNAS,)108,)10496' OH U1782 A2602 C2610 A2503 A2059 A2058 Specific tunnel sensors recognize the presence of the stalling cofactor and its structure Vázquez\Laslop)et)al.)(2011),)PNAS,)108,)10496' ✕ Peptidyl transferase The analog signal from the peptide and small molecular cofactor is integrated in the peptidyl transferase center Nascent peptide defines the recognition of the small molecular cofactor MGIFSIFVISTVHYQPNKK 19' ermCL) ermC' MLVFQMRNVDKTSTI… 36' ermB' ermBL) ✔ ✔ Erythromycin (ERY) Telithromycin (TEL) O N N N O O OH HO N OH O O O O O O O O O O O O HO N O OH N O ✔ ✗ O ErmCL: M G I F S I F V I . . . . . . ! ! ! ! ErmBL: M L V F Q M R N V D . . . . .! ! ! ! CL-BL: M G I F S M R N V D . . . . . ! ! ' ERY TEL ✔ ✗ ✔ ✔ ✔ ✔ The potential to discriminate between the cofactor structures resides in the C-terminus of the stalling nascent peptide Erythromycin Telithromycin F Y N G Q E T stalling P P WW M L V F Q M R N V D A R C H I L K M S V F Y A R C H I L K M S V N G Q E T no stalling ErmBL: M L V F Q M R N V D . . . . .! Telithromycin Stalling peptide R N V D! Ery Tel O N N N +'' +' R N V E! +' 0' R N V Y! 0' +' R N V A! 0' 0' Erythromycin O O OH HO OH N O O O O O O O O O O O O O HO N O N OH O The nature of the P-site amino acid defines what small molecule can be recognized as a stalling cofactor. Small molecule can affect the discriminating properties of the ribosome peptidyl-tRNA drop-off Macrolides stop protein synthesis at the early rounds of translation by promoting drop-off of peptidyl-tRNAs with short nascent peptides No'an)bio)c' Erythromycin' Telithromycin' O N OH HO N N OH O O N O O O O O O O O O O HO O N OH O O O N O Specific proteins can be synthesized y the drug-bound ribosome. The structure of the antibiotic in the tunnel affects the spectrum of synthesized proteins Kannan)et)al.)(2012),)Cell,)151,)508' ! Специальные нуклеотидные сеносры в туннеле позволяют рибосоме «узнавать» определенные структуры в синтезируемом белке. ! Антибиотик помогает рибосоме распознать такие структуы, но и сам антибиотик распознается как сигнал остановки трансляции. ! Меняя структуру белка, закодированого в мРНА можно научить рибосому узнавать разные низкомолекулярнуе соединения. ! Таким образом рибосома выступает в качестве сенсора и вновь синтесируемого пептида и некоторых малых молекул [email protected] Nora Vazquez-Laslop Mashal Almutairi Krishna Kannan Dorota Klepacki Pulkit Gupta Teresa Szal Collaborations: Tanel Tenson (Tartu University) Ada Yonath (Weizmann Institute) Jonathan Weissman (UCSF) Priya Sothiselvam Tom Steitz (Yale) Daniel Wilson (U of Munich) Klaus Schulten (U of Illinois)